A Multicomponent Animal Virus Isolated from Mosquitoes Jason T. Ladner, Michael R. Wiley, Brett Beitzel, Albert J. Auguste, Alan P. Dupuis, Michael E. Lindquist, Samuel D. Sibley, Krishna P. Kota, David Fetterer, Gillian Eastwood, David Kimmel, Karla Prieto, Hilda Guzman, Matthew T. Aliota, Daniel Reyes, Ernst E. Brueggemann, Lena St. John, David Hyeroba, Michael Lauck, Thomas C. Friedrich, David H. O’Connor, Marie C. Gestole, Lisa H. Cazares, Vsevolod L. Popov, Fanny Castro-Llanos, Tadeusz J. Kochel, Tara Kenny, Bailey White, Michael D. Ward, Jose R. Loaiza, Tony L. Goldberg, Scott C. Weaver, Laura D. Kramer, Robert B. Tesh, Gustavo Palacios  Cell Host & Microbe  Volume 20, Issue 3, Pages 357-367 (September 2016) DOI: 10.1016/j.chom.2016.07.011 Copyright © 2016 Elsevier Inc. Terms and Conditions

Cell Host & Microbe 2016 20, 357-367DOI: (10.1016/j.chom.2016.07.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 The Genome of GCXV Is Enveloped and Divided into Five Single-Stranded Positive-Sense RNA Segments (A) Distinct RNA segments of GCXV on an agarose gel. Segment numbers are shown in black; ladder band sizes (nt) are shown in white. (B) Selective degradation with RNase I demonstrates that GCXV has a single-stranded RNA genome. U, untreated; D, DNase I treated; R, RNase I treated. Segment 4 amplicons were run on a separate gel. (C) Genome schematic with all ORFs ≥ 400 nt. Dotted lines indicate regions putatively translated through −1 ribosomal frameshifting (arrows indicate slippery heptanucleotides); solid lines indicate the predicted ORFs based on the first conserved AUG. (D) Treatment with NP40 resulted in highly diminished RNA copy numbers, lack of growth, and absence of CPE in C6/36 cells, indicating that GCXV is enveloped. See also Figures S2 and S3 and Tables S2 and S3. Cell Host & Microbe 2016 20, 357-367DOI: (10.1016/j.chom.2016.07.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Segment 5 of GCXV Is Not Required for Replication in C6/36 Cells and Is Variably Present in Natural Isolates (A) Cell culture supernatants resulting from transfection with GCXV segments 1–5 (passage 1) and segments 1–4 (passage 2). Segment numbers are shown in black; ladder band sizes (nt) are shown in white. (B) Unrooted, nt-level phylogenetic trees including all six isolates of GCXV. ACH27 and TR7094 lack segment 5. Color indicates country of collection: blue for Peru, red for Panama, and green for Trinidad. Branch labels represent bootstrap support values. The scale bar indicates the number of nt changes per site. See also Table S4. Cell Host & Microbe 2016 20, 357-367DOI: (10.1016/j.chom.2016.07.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 The Dose-Response Curve for GCXV Supports a Multicomponent Genome Organization Plaque count is shown on the y axis, and the relative dilution of the virus stock is shown on the x axis. The analysis included five replicates per dilution (circles). The black line is the best-fitting line from a Poisson generalized linear model, with 95% confidence intervals (gray band). The gray lines represent expected slopes for multiplicities of infection of 1–5. Cell Host & Microbe 2016 20, 357-367DOI: (10.1016/j.chom.2016.07.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Multiple Combinations of GCXV Genome Segments Were Detected in Infected Cells A representative 63× magnification image is shown from the co-hybridization of probes for segments 3–5. Four different infected cell types were observed: closed arrowhead, missing segments 4 and 5; open arrowhead, missing segment 5; full arrow, missing segment 4 (note that segment 5 present in low abundance in the indicated cell); and dashed arrow, containing all assayed segments. The scale bar represents 10 μm. See also Figure S1 and Table S1. Cell Host & Microbe 2016 20, 357-367DOI: (10.1016/j.chom.2016.07.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 GCXV Belongs to an Insect-Specific Clade within the Segmented Jingmenviruses Maximum likelihood (ML) trees are shown with bootstrap support values from both the ML and neighbor-joining (NJ) trees: ML/NJ. The scale indicates the number of amino acid changes per site. See also Table S5. Cell Host & Microbe 2016 20, 357-367DOI: (10.1016/j.chom.2016.07.011) Copyright © 2016 Elsevier Inc. Terms and Conditions